Study On The Role And Mechanism Of Ferroptosis In Renal Tubular Epithelial Cell Injury Induced By Calcium Oxalate Crystals

Objective:To intervene renal tubular epithelial cells（HK-2）through calcium oxalate（CaOx）crystals,and to explore the mechanism of cell injury induced by CaOx crystals.And using ferroptosis inducers and inhibitors to explore the mechanism of ferroptosis in our renal tubular epithelial cell-calcium oxalate crystals（HK-2-CaOx）reaction model,and analyze whether it can be regulated byferroptosis To play a regulatory role in HK-2 cell damage induced by CaOx crystals.Method:Prepared CaOx crystal suspension was used to intervene HK-2 cells to construct HK-2-CaOx reaction model,using 7 groups of different concentrations of CaOx crystals（0,0.25,0.5,1.0,2.0,4.0,8.0 mmol/L）Intervene in HK-2 cells for 24 hours,extract HK-2 cell protein after the intervention,and use CaOx crystals at a concentration of 4.0 mmol/L to intervene in HK-2 cells at different time points（0,3,6,9,12,24,48 h）extract cell protein,and detect the expression of ferroptosis marker protein glutathione peroxidase 4（GPX4）in each group by Western blotting.At the same time,the ferroptosis inducer Erastin and the ferroptosis inhibitor Ferrostatin-1（Fer-1）were used to intervene in HK-2 cells for 24 hours to regulate the level of intracellular ferroptosis.According to the requirements of the experimental design,HK-2 cells were randomly divided into 4 groups:normal control group（NC;no intervention treatment,cultured with complete medium only）;calcium oxalate crystal stimulation group（CaOx;use containing 4.0 mmol/L Complete medium culture of CaOx crystals）;calcium oxalate crystals+Erastin treatment group（CaOx+Erastin;cultured in complete medium containing 10.0 umol/L Erastin and 4.0 mmol/L CaOx crystals）;calcium oxalate crystals+Ferrostatin-1treatment Group（CaOx+Fer-1;use a complete medium containing 1.0 umol/L Fer-1 and 4.0 mmol/L CaOx crystals）.After 24 hours,Western blotting,real-time fluorescent quantitative PCR（RT-q PCR）and immunofluorescence techniques were used to analyze the ferroptosis-related protein GPX4,long-chain fatty acyl-Co A synthase 4（ACSL4）and solute carrier family 7member 11（SLC7A11）Expression in renal tubular epithelial cells;Use the transmission electron microscope to observe the morphological changes of the organelles;use the kit to detect the contents of malondialdehyde（MDA）and glutathione（GSH）in HK-2 cells;use the total superoxide dismutase（SOD）enzyme kit to detect Enzyme activity of total superoxide dismutase in renal tubular epithelial cells;DCFH-DA fluorescent staining method was used to observe the expression of cellular reactive oxygen species（ROS）in renal tubular epithelial cells,and the above method was used to explore the role of ferroptosis in the cell-crystal reaction model Mechanism and role.The adhesion of CaOx in HK-2 cells of each group was observed by optical microscope,the expression level of LDH in the cell supernatant was detected by the lactate dehydrogenase（LDH）kit,and the nuclear damage of HK-2 cells was detected by DAPI staining.To explore the regulatory role of ferroptosis in renal tubular epithelial cell injury induced by calcium oxalate crystals.Results:During the intervention of CaOx crystals in HK-2 cells,as the concentration and time of CaOx crystal intervention increased,the expression level of ferroptosis marker protein GPX4 gradually decreased.The experimental results showed that compared with the control group,4mmol/L,The intervention concentration of calcium oxalate crystals of 8mmol/L was statistically significant（P<0.05）.Compared with the control group,the 4 mmol/L calcium oxalate crystal stimulation group（P<0.05）showed a significant difference in the expression of the intracellular ferroptosis-related protein GPX4 after 24 hours of exposure to the cells.Therefore,4 mmol/L calcium oxalate crystals were used as the best Concentration interferes with cells.In addition,compared with the control group,the intervention time was statistically significant at 24h and 48h（P<0.05）.Among them,the 24h intervention group（P<0.05）had obvious differences in the expression of GPX4.Therefore,24h was selected as the optimal intervention time for calcium oxalate.After using the ferroptosis inducer Erastine and the ferroptosis inhibitor Ferrostatin-1 to interfere with renal tubular epithelial cells,the level of ferroptosis induced by calcium oxalate crystals changed significantly.Compared with the normal control group,CaOx crystals induced HK-2 The expression of GPX4 and SLC7A11 protein in cells was significantly reduced（P<0.05）,and the expression of ACSL4 protein was significantly increased（P<0.05）.After adding the ferroptosis protection agent Fer-1,this result was significantly inhibited.Compared with the calcium oxalate crystal group alone,after adding calcium oxalate crystals and ferroptosis protection agent Fer-1 at the same time,the protein expression of GPX4 and SLC7A11 increased（P<0.05）,the expression of ACSL4 protein was significantly reduced（P<0.05）.Compared with the simple calcium oxalate crystal group,after adding calcium oxalate crystal and Erastin at the same time,the protein expression of GPX4 and SLC7A11 was significantly reduced（P<0.05）,and the protein expression of ACSL4 was significantly increased（P<0.05）.The RT-q PCR results were consistent with the protein detection results.The experimental results showed that the expression of ferroptosis-related protein GPX4 m RNA in the calcium oxalate crystal stimulation group was significantly lower than that of the normal control group（P<0.05）.The expression level of ferroptosis marker protein GPX4 m RNA in the calcium oxalate crystals+ferroptosis protector Fer-1 intervention group was significantly higher than that of the calcium oxalate crystals group（P<0.05）,while the calcium oxalate crystals+ferroptosis inducer Erastin intervention group was higher than the oxalic acid The m RNA expression level of the ferroptosis marker protein GPX4 in the calcium crystal group was significantly reduced（P<0.05）.At the same time,compared with the normal control group,the calcium oxalate crystal stimulation group had a significantly higher m RNA expression level of the ferroptosis-related protein ACSL4（P<0.05）.Compared with the calcium oxalate crystals group,the expression level of ACSL4 m RNA of the ferroptosis marker egg in the calcium oxalate crystals+ferroptosis protector Fer-1 intervention group was significantly lower（P<0.05）,while the calcium oxalate crystals+ferroptosis inducer Erastin intervention group was lower than the oxalic acid The expression level of the ferroptosis marker protein ACSL4 m RNA in the calcium crystal group was significantly increased（P<0.05）.In the calcium oxalate crystal stimulation group,the contents of superoxide dismutase and glutathione were significantly reduced（P<0.05）,the content of malondialdehyde was significantly increased（P<0.05）,and the fluorescence intensity of intracellular ROS increased（P<0.05）.The level of oxidative stress in tubular epithelial cells is positively correlated with the level of ferroptosis.At the same time,transmission electron microscopy observed significant changes in the morphology of mitochondria in HK-2 cells after the intervention of calcium oxalate crystals.Compared with the normal control group,the calcium oxalate crystal stimulation group had smaller intracellular mitochondria and increased mitochondrial membrane density.And some mitochondrial cristae are reduced or disappeared^[1].Ferrostatin-1 treatment can significantly reduce the CaOx crystal-induced HK-2 cell crystal adhesion and cell nucleus damage,and reduce the secretion of lactate dehydrogenase in HK-2cells（P<0.05）,while Erastin treatment further aggravated HK-2 cells The adhesion of CaOx crystals,the damage of cell nucleus and the secretion of lactate dehydrogenase（P<0.05）.In conclusion:By constructing a cell-crystal reaction model,the optimal concentration and time for CaOx crystals to interfere with the iron death reaction of HK-2 cells were determined.CaOx crystals can induce ferroptosis in HK-2cells by increasing the level of oxidative stress in HK-2 cells.The application of the classic ferroptosis inducer Erastin and the inhibitor Ferrostatin-1 can effectively regulate the oxidative stress level in renal tubular epithelial cells,thereby regulating the occurrence of iron death of renal tubular epithelial cells.Ferrostatin-1 treatment can significantly reduce the occurrence of iron death in renal tubular epithelial cells,and significantly reduce the cell damage induced by CaOx crystals,while treatment with Erastin further aggravates the occurrence of iron death in HK-2 cells.After the intervention of CaOx crystals,cell damage The damage also worsened.